Compositions and methods for treating ischemic heart disease

ABSTRACT

The present disclosure is directed to the treatment of ischemic heart disease and clinical conditions associated with ischemic heart disease. A composition containing a monoclonal antibody directed against gastric inhibitory polypeptide is administered. This results in cardioprotective effects against acute myocardial infarction, such as a decrease in circulating triglycerides, total cholesterol, and low-density lipoproteins, and an increase in the ratio of high-density lipoprotein to total cholesterol.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/507,436, filed Jul. 10, 2019, now U.S. Pat. No. 11,066,467, whichclaims priority to U.S. Provisional Patent Application Ser. No.62/696,222, filed Jul. 10, 2018. U.S. patent application Ser. No.16/507,436 is also a continuation-in-part of U.S. patent applicationSer. No. 15/714,440, filed Sep. 25, 2017, which is a continuation ofU.S. patent application Ser. No. 14/573,600, filed on Dec. 17, 2014, nowU.S. Pat. No. 9,771,422; which claimed priority to U.S. ProvisionalPatent Application Ser. No. 61/917,136, filed on Dec. 17, 2013; to U.S.Provisional Patent Application Ser. No. 61/974,660, filed on Apr. 3,2014; to U.S. Provisional Patent Application Ser. No. 62/007,255, filedon Jun. 3, 2014; to U.S. Provisional Patent Application Ser. No.62/045,189, filed on Sep. 3, 2014; to U.S. Provisional PatentApplication Ser. No. 62/074,225, filed on Nov. 3, 2014; to U.S.Provisional Patent Application Ser. No. 62/074,227, filed on Nov. 3,2014; and to U.S. Provisional Patent Application Ser. No. 62/074,234,filed on Nov. 3, 2014. The disclosures of these applications are fullyincorporated by reference herein.

BACKGROUND

A sequence listing is being submitted herein as an ASCII text file withthe name “LOBE200009US01_11Mar2021_SeqListing.txt”, created on Mar. 11,2021, with a file size of 23,874 bytes. The material in this text fileis hereby fully incorporated by reference herein.

The present disclosure relates to compositions and methods for treatingischemic heart disease using monoclonal antibodies (mAbs) that bind toglucose-dependent insulinotropic polypeptide, also known as gastricinhibitory polypeptide (GIP).

Ischemic heart disease, also referred to as coronary heart disease orIHD or CHD, occurs when a patient has one or more symptoms, signs, orcomplications from an inadequate supply of blood to the myocardium. Thisis most commonly due to obstruction of the coronary arteries due toatherosclerosis. The care of patients with ischemic heart diseaseincludes ascertainment of the diagnosis and its severity, control ofsymptoms, and therapies to improve survival.

Stable angina pectoris (angina) is characterized by chest discomfortthat occurs predictably and reproducibly at a certain level of exertionand is relieved with rest or nitroglycerin treatment or othermedications that improve coronary circulation. Angina occurs whenmyocardial oxygen demand exceeds oxygen supply. Most patients withischemic heart disease will experience angina as part of the clinicalmanifestations of the disease. During the advanced stages of ischemicheart disease, angina may occur at rest, at which time it is designatedas unstable angina. Many patients can be given the diagnosis of ischemicheart disease based on a history of angina pectoris in the presence ofone or more risk factors for atherosclerotic cardiovascular disease.

Risk factors for the development of ischemic heart disease arewell-recognized and include hypertension, tobacco, use, obesity,hyperlipidemia, diabetes mellitus, and family history. With theexception of family history, these risk factors are modifiable.Addressing these risk factors should encompass a central component ofthe management of patients with angina. Such measures include thetreatment of hypertension, cessation of smoking, initiation of statintherapy, weight reduction, glycemic control in diabetics, andparticipation in regular physical activity.

Despite changes aimed at risk reduction, ischemic heart diseasegenerally progresses, and patients may develop acute coronary syndrome(ACS). ACS comprises a set of signs and symptoms that are due to markedreductions in coronary artery blood flow, resulting in damage or evendeath of the part of affected heart muscle. ACS is commonly associatedwith three clinical manifestations: ST elevation myocardial infarction(STEMI), non-ST elevation MI (NSTEMI), or unstable angina.

The survival rate for American patients hospitalized with acutemyocardial infarction (MI) is approximately 95%, which representssignificant improvement in survival and is related to advances inemergency medical response and treatment strategies. The incidence of MIincreases with age; however, the actual incidence is dependent onpredisposing risk factors for atherosclerosis. Approximately 50% of allMIs in the United States occur in people younger than 65 years of age.However, as demographics shift and the mean age of the populationincreases, a larger percentage of patients presenting with MI willlikely be greater than 65 years of age.

The therapeutic goals of acute MI are the expeditious restoration ofnormal coronary blood flow and the maximum salvage of functionalmyocardium. These goals can be accomplished by several medicalinterventions and adjunctive therapies. The principal impediments toachieving these goals are the patient's failure to recognize symptomsquickly, which leads to a delay in seeking medical attention. Whenpatients present to a hospital, a variety of interventions are availableto diminish morbidity and mortality. General medical therapy includesthe use of antiplatelet agents such as aspirin and clopidogrel, as wellas nitroglycerine, supplemental oxygen, beta-blockers, and pain control,generally in the form of morphine sulfate.

Additionally, angiotensin-converting enzyme (ACE) inhibitors should beused in all patients with a STEMI without contraindications. ACEinhibitors are also recommended in patients with NSTEMI who havediabetes, heart failure, hypertension, or evidence of compromisedcardiac ventricular function characterized by an ejection fraction ofless than 40%. Other agents that are used in the acute MI includeunfractionated heparin, low-molecular-weight heparin (LMWH), warfarin,glycoprotein Ilb/Illa receptor antagonists, statins, and aldosteroneantagonists.

In the case of ACS, both pharmacological and invasive methods areemployed to restore normal coronary blood flow. The former includesfibrinolytic therapy which is utilized for patients who present with aSTEMI within 12 hours of symptom onset provided they have nocontraindication to its use. These drugs are plasminogen activators andhave been shown to restore normal coronary blood flow in 50%-60% ofSTEMI patients. A fibrinolytic agent is most effective within the firsthour of symptom onset, and in particular, within 30 minutes of symptomonset.

Invasive methods used to restore coronary blood flow includepercutaneous coronary intervention (PCI), which consists of diagnosticangiography combined with angioplasty and generally stenting. Bare metalor drug-eluting stents are employed. Emergency PCI is more effectivethan fibrinolytic therapy in facilities that offer PCI by experiencedpersonnel when performed in a timely fashion. Patients with STEMI shouldhave PCI within 90 minutes of arrival at the hospital if skilled cardiaccatheterization services are available. Patients with NSTEMI and varioushigh-risk features are recommended to undergo PCI within 48 hours ofsymptom presentation. When performed by skilled personnel, PCI cansuccessfully restore coronary blood flow in 90%-95% of MI patients.

Although its use has diminished in recent years because of the increaseduse and success of PCI, emergency surgery consisting of coronary arterybypass grafting (CABG) is still employed. CABG is warranted in thesetting of failed PCI in patients with hemodynamic instability, as wellas in the setting of mechanical complications of MI and in the settingof mechanical complications of MI provided the coronary anatomy isamenable to such surgical intervention. Restoration of coronary bloodflow with emergency CABG can limit myocardial injury and cell death ifperformed within 3 hours of symptom onset.

Despite the aforementioned advances in care of patients with ischemicheart disease, approximately 450,000 people die from ischemic heartdisease each year in the United States. Thus, the optimal treatment forthese patients remains risk reduction. It would be desirable to developcompositions that mitigate risk factors for the development andprogression of ischemic heart disease. Additionally, methods for suchmitigation are also desired.

BRIEF SUMMARY OF THE DISCLOSURE

The present disclosure is directed to monoclonal antibodies (mAbs) thatbind to gastric inhibitory polypeptide (GIP), also known asglucose-dependent insulinotropic polypeptide, and to methods of usingsuch mAbs to treat ischemic heart disease, also known as coronary arterydisease.

Disclosed herein in various embodiments are methods of treating severaldifferent conditions or manifestations associated with ischemic heartdisease. Such conditions or manifestations may include stable anginapectoris (angina), unstable angina, and acute coronary syndrome (ACS).ACS includes three clinical scenarios: ST elevation myocardialinfarction (STEMI); non-ST elevation MI (NSTEMI); and unstable angina.

Disclosed herein in various embodiments are methods of treating ischemicheart disease or clinical manifestations thereof, comprisingadministering to a person a composition comprising a pharmaceuticallyeffective amount of a molecular antagonist of GIP to reduce myocardialinfarction (MI)-induced injury to the heart and to enhance survival.

The molecular antagonist comprises at least one complementaritydetermining region (CDR) with at least 80% identity to an amino acidsequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 31, SEQ IDNO: 32, and SEQ ID NO: 33.

In specific embodiments, the molecular antagonist comprises a lightchain variable domain having a first CDR and a second CDR, each CDRhaving at least 95% identity to an amino acid sequence selected from thegroup consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IDNO: 23, and SEQ ID NO: 24. The first CDR and the second CDR of themolecular antagonist are joined to each other by a linking group. Thelinking group can be a chain of amino acids.

In other embodiments, the molecular antagonist comprises a light chainvariable domain having a first CDR with at least 80% identity to theamino acid sequence of SEQ ID NO: 20, a second CDR with at least 80%identity to the amino acid sequence of SEQ ID NO: 21, and a third CDRwith at least 85% identity to the amino acid sequence of SEQ ID NO: 22.The first CDR, the second CDR, and the third CDR of the molecularantagonist are joined to each other by linking groups. The linkinggroups can be independently a chain of amino acids.

In still other embodiments, the molecular antagonist comprises a lightchain variable domain having at least 80% identity to an amino acidsequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, and SEQ ID NO: 19.

In yet still more embodiments, the molecular antagonist comprises aheavy chain variable domain having a first CDR with at least 80%identity to the amino acid sequence of SEQ ID NO: 31, a second CDR withat least 80% identity to the amino acid sequence of SEQ ID NO: 32, and athird CDR with at least 80% identity to the amino acid sequence of SEQID NO: 33.

In some embodiments, the molecular antagonist comprises a heavy chainvariable domain having at least 80% identity to an amino acid sequenceselected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQID NO: 29, and SEQ ID NO: 30.

In particular embodiments, the molecular antagonist comprises a lightchain variable domain and a heavy chain variable domain; wherein thelight chain variable domain comprises a first CDR and a second CDR, eachCDR having at least 95% identity to an amino acid sequence selected fromthe group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 23, and SEQ ID NO: 24; and wherein the heavy chain variabledomain comprises a first CDR with at least 80% identity to the aminoacid sequence of SEQ ID NO: 31, a second CDR with at least 80% identityto the amino acid sequence of SEQ ID NO: 32, and a third CDR with atleast 80% identity to the amino acid sequence of SEQ ID NO: 33. Themolecular antagonist can be a single-chain variable fragment (scFv), anF(ab′)2 fragment, a Fab or Fab′ fragment, a diabody, a triabody, atetrabody, or a monoclonal antibody.

In other embodiments, the molecular antagonist comprises a light chainvariable domain and a heavy chain variable domain; wherein the lightchain variable domain has at least 80% identity to an amino acidsequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, and SEQ ID NO: 19; and wherein the heavy chainvariable domain has at least 80% identity to an amino acid sequenceselected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQID NO: 29, and SEQ ID NO: 30. The molecular antagonist can be asingle-chain variable fragment (scFv), an F(ab′)2 fragment, a Fab orFab′ fragment, a diabody, a triabody, a tetrabody, or a monoclonalantibody.

In specific embodiments, the molecular antagonist is a monoclonalantibody with a light chain variable domain having at least 80% identityto SEQ ID NO: 18, and a heavy chain variable domain having at least 80%identity to an amino acid sequence selected from the group consisting ofSEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30.

In more specific embodiments, the molecular antagonist is a wholemonoclonal antibody with a light chain variable domain having at least90% identity to SEQ ID NO: 18, and a heavy chain variable domain havingat least 90% identity to SEQ ID NO: 29.

The molecular antagonist may bind to an amino acid sequence of GIP, theamino acid sequence being selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

In particular embodiments, the molecular antagonist is a wholemonoclonal antibody comprising human constant regions.

The molecular antagonist may have a MW of about 30 kDa to about 500 kDa.The molecular antagonist may have a binding affinity for GIPcharacterized by an IC₅₀ of about 0.1 nM to about 7 nM.

The composition can be administered intravenously, intraperitoneally, orsubcutaneously. The composition can further comprise an inertpharmaceutical excipient selected from the group consisting of bufferingagents, surfactants, preservative agents, bulking agents, polymers, andstabilizers. The composition may be in the form of a powder, injection,solution, suspension, or emulsion.

The composition may contain the monoclonal antibody antagonist in anamount of from about 0.1 to about 1000 milligram per milliliter of thecomposition. Sometimes, the composition is lyophilized.

Also disclosed herein are molecular antagonists of gastric inhibitorypolypeptide (GIP), which can take several forms such as whole monoclonalantibodies and variants thereof. The molecular antagonists are asdescribed above.

Also disclosed herein are complementary DNA sequences having at least85% identity to a DNA sequence selected from the group consisting of SEQID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39,SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42.

These and other non-limiting features of the present disclosure arediscussed in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawings, which arepresented for the purposes of illustrating the exemplary embodimentsdisclosed herein and not for the purposes of limiting the same.

C57BL/6 mice fed a high-fat diet are an established model ofdiet-induced obesity and associated metabolic syndrome. The“HFD-control” mice were fed a high-fat diet (HFD) and administeredphosphate buffered saline (PBS) by intraperitoneal injection. The“HFD-mAb” mice were fed a HFD diet and administered, by intraperitonealinjection, the monoclonal antibody antagonist of gastric inhibitorypolypeptide (GIP mAb) in PBS at 60 mg/kg body weight (BW) per week. The“Iso-diet” mice were fed a low-fat isocaloric diet and not treated. Inthe HFD diet, about 60% of total calories come from fat, while in theisocaloric diet, about 10% of total calories come from fat.

FIG. 1 is a bar graph showing triglyceride (TG) levels measured in thesera of the three groups of mice. At the end of the 17-week diet period,mice were sacrificed, whole blood was collected, serum was prepared andserum triglycerides measured. The average serum TG levels for the micein each group are plotted. TG levels in HFD-control mice were 1.44 timesgreater than TG levels in HFD-mAb mice (p=0.02). Lower TG levels arebetter. The y-axis is in units of mg/dL, and runs from 0 to 100 atincrements of 10.

FIG. 2 is a bar graph showing total cholesterol (TC) levels measured inthe sera of the three groups of mice after the 17-week diet period. TClevels in HFD-control mice were 1.55 times greater than TC levels inHFD-mAb mice (p=0.006). Lower TC levels are better. The y-axis is inunits of mg/dL, and runs from 0 to 300 at increments of 50.

FIG. 3 is a bar graph showing the ratio of high-density lipoprotein(HDL) to total cholesterol (TC) measured in the sera of the three groupsof mice after the 17-week diet period. The average value for each groupis plotted. The ratio of HDL to total cholesterol in HFD-mAb mice was25% higher than the ratio of HDL to total cholesterol levels inHFD-control mice (p value=0.03). Higher ratios of HDL to totalcholesterol levels are desirable. The y-axis is the HDL/TC ratio, andruns from 0 to 0.5 at increments of 0.05.

FIG. 4 is a bar graph showing low-density lipoprotein (LDL) levelsmeasured in the sera of the three groups of mice after the 17-week dietperiod. The average serum LDL levels for the mice in each group areplotted. LDL levels in HFD-control mice were 1.95 times greater than LDLlevels in HFD-mAb mice (p=0.007). Lower LDL (the “bad” cholesterol)levels are better. The y-axis is in units of mg/dL, and runs from 0 to120 at increments of 20.

FIG. 5 is a graph showing the relative activity of three humanizedantibodies for GIP, along with the original antibody, a positivecontrol, and a negative control, in an in vitro neutralization assay.The y-axis is the relative activity, and is unitless, and runs from 0 to0.12 at increments of 0.02.

FIG. 6 is a graph showing the amount of anti-GIP antibody in plasma overtime for control mice and “mAb” mice. The y-axis is in units ofmicrograms per milliliter (pg/mL), and runs from 0 to 40 in incrementsof 5. The x-axis is time in hours, and runs from 0 to 350 hours inincrements of 50. The top line (dashed) is for the “mAb” mice, and thebottom line (solid) is for the control mice.

DETAILED DESCRIPTION

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise.

As used in the specification and in the claims, the open-endedtransitional phrases “comprise(s),” “include(s),” “having,”“contain(s),” and variants thereof require the presence of the namedingredients/steps and permit the presence of other ingredients/steps.These phrases should also be construed as disclosing the closed-endedphrases “consist of” or “consist essentially of” that permit only thenamed ingredients/steps and unavoidable impurities, and exclude otheringredients/steps.

Numerical values in the specification and claims of this applicationshould be understood to include numerical values which are the same whenreduced to the same number of significant figures and numerical valueswhich differ from the stated value by less than the experimental errorof conventional measurement technique of the type described in thepresent application to determine the value.

All ranges disclosed herein are inclusive of the recited endpoint andindependently combinable (for example, the range of “from 2 grams to 10grams” is inclusive of the endpoints, 2 grams and 10 grams, and all theintermediate values).

The term “about” can be used to include any numerical value that canvary without changing the basic function of that value. When used with arange, “about” also discloses the range defined by the absolute valuesof the two endpoints, e.g. “about 2 to about 4” also discloses the range“from 2 to 4.” The term “about” may refer to plus or minus 10% of theindicated number.

The term “identity” refers to the similarity between a pair of sequences(nucleotide or amino acid). Identity is measured by dividing the numberof identical residues by the total number of residues and multiplyingthe product by 100 to achieve a percentage. Thus, two copies of exactlythe same sequence have 100% identity, while sequences that havedeletions, additions, or substitutions may have a lower degree ofidentity. Those skilled in the art will recognize that several computerprograms, such as those that employ algorithms such as BLAST, areavailable for determining sequence identity. BLAST nucleotide searchesare performed with the NBLAST program, and BLAST protein searches areperformed with the BLASTP program, using the default parameters of therespective programs.

Two different sequences may vary from each other without affecting theoverall function of the protein encoded by the sequence. In this regard,it is well known in the art that chemically similar amino acids canreplace each other, often without change in function. Relevantproperties can include acidic/basic, polar/nonpolar, electrical charge,hydrophobicity, and chemical structure. For example, the basic residuesLys and Arg are considered chemically similar and often replace eachother, as do the acidic residues Asp and Glu, the hydroxyl residues Serand Thr, the aromatic residues Tyr, Phe and Trp, and the non-polarresidues Ala, Val, Ile, Leu and Met. These substitutions are consideredto be “conserved.”

For purposes of the present disclosure, when comparing amino acidsequences for % identity, any deletions will only occur at the ends ofthe amino acid sequences, and not in the middle of the sequence. Also,for purposes of the present disclosure, the following seven groups ofamino acids are considered to be conservative substitutions with eachother (cysteine and proline have no conservative substitutions) whendetermining % identity:

Cysteine (C) Proline (P) Valine, isoleucine, leucine, methionine,alanine, glycine (V, I, L, M, A, G) Histidine, lysine, arginine (H, K,R) Phenylalanine, tryptophan, tyrosine (F, W, Y) Serine, threonine,asparagine, glutamine (S, T, N, Q) Aspartic acid, glutamic acid (D, E)

Similarly, nucleotide codons and acceptable variations are known in theart. For example, the codons ACT, ACC, ACA, and ACG all code for theamino acid threonine, i.e. the third nucleotide can be modified withoutchanging the resulting amino acid. Similarity is measured by dividingthe number of similar residues by the total number of residues andmultiplying the product by 100 to achieve a percentage. Note thatsimilarity and identity measure different properties.

An “antibody” is a protein used by the immune system to identify atarget antigen. The basic functional unit of an antibody is animmunoglobulin monomer. The monomer is made up of two identical heavychains and two identical light chains which form a Y-shaped protein.Each light chain is composed of one constant domain and one variabledomain. For light chains, the constant domain may also be referred to asthe “constant region”, and the variable domain can also be referred toas the “variable region”. Each heavy chain is composed of one variabledomain and three or four constant domains. For heavy chains, theconstant domains together are referred to as the “constant region”, andthe variable domain can also be referred to as the “variable region”.The arms of the Y are called the fragment, antigen-binding (Fab) region,with each arm being called a Fab fragment. Each Fab fragment is composedof one constant domain and one variable domain from a heavy chain, andone constant domain and one variable domain from a light chain. The baseof the Y is called the Fc region, and is composed of two or threeconstant domains from each heavy chain. The variable domains of theheavy and light chains in the Fab region are the part of the antibodythat binds to GIP. More specifically, the complementarity determiningregions (CDRs) of the variable domains bind to their antigen (i.e. theGIP). In the amino acid sequence of each variable domain, there arethree CDRs arranged non-consecutively. The term “whole” is used hereinto refer to an antibody that contains the Fab region and the Fc region.

An “antagonist of GIP” according to the present disclosure is a moleculethat binds to GIP and interferes with the biological action of GIP.

The present disclosure relates to methods of treating patients withmolecules that antagonize GIP, i.e. bind to GIP. In this regard,glucose-dependent insulinotropic polypeptide, also referred to asgastric inhibitory polypeptide or GIP, is an insulinotropic peptidereleased from intestinal K-cells during the postprandial period. As anincretin, GIP stimulates insulin secretion by stimulating pancreaticbeta cells in response to food intake. GIP, also notated herein asGIP(1-42), primarily circulates as a 42-amino acid polypeptide, but isalso present in a form lacking the first 2 N-terminal amino acids(GIP(3-42)). GIP(1-30)-NH₂ or GIP(1-30)-alpha amide is a syntheticderivative of GIP(1-42) that lacks the last 12 C-terminal amino acids.GIP(1-30)-NH₂ has the same biological functions as GIP(1-42). Naturallyoccurring GIP(1-30)-NH₂ has been hypothesized, but has not beenidentified with certainty in any biological species.

GIP functions via binding to its cognate receptor (GIPR) found on thesurface of target cells. GIPR is a member of the glucagon-secretinfamily of G-protein coupled receptors (GPCRs), possessing seventransmembrane domains. Native GIP(1-42) and the synthetic derivativeGIP(1-30)-NH₂ bind to GIPR with high affinity and possess agonistproperties. Native GIP(1-42) and the synthetic derivative GIP(1-30)-NH₂also inhibit lipolysis in adipocytes induced by glucagon andβ-adrenergic receptor agonists, including isoproterenol.

GIP is well-conserved between humans (Homo sapiens) (SEQ ID NO: 1), mice(Mus musculus) (SEQ ID NO: 2), rats (Rattus norvegicus) (SEQ ID NO: 3),and pigs (Sus scrofa) (SEQ ID NO: 4). There are only four substitutionsbetween the four sequences, all of which are conserved. The 42-aminoacid sequences from these four species are listed below:

SEQ ID NO: 1: YAEGT FISDY SIAMD KIHQQ DFVNW LLAQK GKKND WKHNI TQSEQ ID NO: 2: YAEGT FISDY SIAMD KIRQQ DFVNW LLAQR GKKSD WKHNI TQSEQ ID NO: 3: YAEGT FISDY SIAMD KIRQQ DFVNW LLAQK GKKND WKHNL TQSEQ ID NO: 4: YAEGT FISDY SIAMD KIRQQ DFVNW LLAQK GKKSD WKHNI TQ

The present application relates to methods of treating ischemic heartdisease, as well as diseases, disorders and conditions associated withthe development and progression of ischemic heart disease usingcompositions containing molecular antagonists that bind to GIP. Inparticular embodiments, the molecular antagonists are whole monoclonalantibodies (i.e. GIP mAbs).

GIP receptors are found throughout the body, including the heart(cardiomyocytes). The present disclosure shows that mAb binding to GIPhas several beneficial properties relating to risk factors for thedevelopment and progression of ischemic heart disease. These beneficialproperties include decreasing circulating triglycerides, totalcholesterol, and low-density lipoproteins (LDL), while increasinghigh-density lipoprotein to total cholesterol (TC) ratio (HDL:TC). It isthus believed that decreasing GIP signaling via mAb binding shouldproduce directly beneficial cardioprotective effects in acute MI. Thiscan be done by administering a composition comprising a GIP monoclonalantibody antagonist to a patient. Other molecular antagonists which arevariations on the CDRs of the monoclonal antibody should also beeffective.

GIP monoclonal antibody antagonists of the present disclosure can begenerated and screened in the following manner. Two peptides with thefollowing amino acid sequences were chemically synthesized:

GIP(1-17)+C: (SEQ ID NO: 5) YAEGT FISDY SIAMD KIC C+mGIP(26-42):(SEQ ID NO: 6) CLLAQ RGKKS DWKHN ITQ

The amino acid sequence of GIP(1-17)+C corresponds to the first 17 aminoacids commonly shared by mature human, mouse, rat, and pig GIP. Thesequence also contains an extra cysteine residue at the N-terminus tofacilitate conjugation to keyhole limpet hemocyanin (KLH). The aminoacid sequence of C+mGIP(26-42) corresponds to the last 17 amino acids ofmature mouse GIP, which has only two substitutions when comparedseparately to human GIP or porcine GIP, and only three substitutionswhen compared to rat GIP. The sequence also contains an extra cysteineresidue at the C-terminus to facilitate conjugation to KLH. The twopeptides were conjugated to KLH. Each conjugate was then used separatelyto inject a group of four mice at four different occasions. Mice wereinjected at weeks 1, 4, 7, 10 and 24 before spleens were harvested.

The spleens were removed from the immunized mice, and splenocytes wereisolated before B cells were fused with immortalized myeloma cells invitro. Polyethylene glycol (PEG) was included in the fusion process toincrease efficiency. Fused cells or hybridomas were incubated inhypoxanthine-aminopterin-thymidine (HAT) medium for 14 days to killmyeloma cells that did not fuse with B cells. The hybridoma cells werethen diluted with incubation media and transferred to a series of96-well plates. The dilutions were to the extent that each wellcontained approximately one cell. The hybridoma cells were expandedseveral days before conditioned media or supernate was collected forscreening purposes.

96-well plates were coated with synthetic mouse GIP(1-42) (PhoenixPharmaceuticals) using 50 μl of a 4 μg/ml solution in PBS. The hybridomasupernates were then collected and added to the wells containing mouseGIP(1-42) for 4 hours at 37° C. The supernates were then removed andwashed to remove any antibody not bound to mouse GIP. Next, a solutionof goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) wasadded to the wells. The goat anti-mouse IgG-HRP was obtained fromJackson Laboratories and used at a dilution of 1 part per 5000. Afterincubation for 1 hour at 37° C., the antibody solution was removed, andthe wells were washed 2 times with 0.4 mg/ml BSA in PBS.

A solution containing HRP substrate 4 mg/ml o-Phenylenediaminedihydrochloride (OPO) in 0.4 mg/ml urea hydrogen peroxide and 0.05 Mphosphate-citrate, pH 5.0, was then added to each well. To quantify HRPactivity, the absorbance of light at a wavelength of 490 nm for eachwell was measured using an ELISA plate reader. This allowed hybridomasto be identified that generated monoclonal antibodies which would bindto the mouse GIP in the wells.

Next, supernates from those identified hybridomas were subsequentlymixed with a 4 μg/ml solution of mouse GIP for 30 minutes at 37° C.,then added to wells coated with mouse GIP as described above. Afterincubation for 1 hour at 37° C., the wells were washed, and goatanti-mouse IgG-HRP was added to the wells. After incubation for 1 hour,the samples were washed and a solution containing the HRP substrate 4mg/ml OPO in 0.4 mg/ml urea hydrogen peroxide, and 0.05 Mphosphate-citrate, pH 5.0, was added to each well. HRP activity in eachwell was quantified. In this screen, monoclonal antibodies that bound toGIP in suspension would be “neutralized” and would not be able to bindto the GIP fixed in the wells. Therefore, wells with low HRP activitycorresponded to monoclonal antibodies that bound more effectively to GIPin suspension. Using these criteria, five hybridomas were identifiedthat best generated monoclonal antibodies (mAbs) which would bind to themouse GIP in suspension. Due to the high correspondence in identitybetween mouse GIP and human GIP, it was expected that these mAbs whichbound to mouse GIP would also bind to human GIP.

Next, the ability of hybridoma supernates to neutralize GIP and preventligand-receptor interaction, receptor activation and receptor-dependentsignaling was tested using a cell culture system. This system usedreporter cells (LGIPR2 cells), which possess the lacZ gene under thecontrol of a cyclic adenosine monophosphate (cAMP-)-responsive promoterand express the rat GIPR on the cell surface. The addition of GIP tothese cells leads to activation of the GIPR, induction of a signalingcascade that leads to accumulation of cAMP, induction of the lacZ geneand synthesis of β-galactosidase. After the addition of a test sampleand incubation for 4 hours, a colorimetric assay was used to measureβ-galactosidase content in cell lysates. The degree of color change isproportional to the level of β-galactosidase activity. The level ofβ-galactosidase activity is dependent on the amount of free biologicallyactive GIP in the test sample.

Supernates from the five hybridoma clones grown in culture that scoredpositive in the suspension assay were diluted 1:1 and 1:20 withsolutions of mouse or human GIP. The mixtures were then added to LGIPR2cells and incubated before washing and assaying for β-galactosidase.Three of the five supernates showed significant inhibition of mouse GIPwhen diluted 1:1, and two of those three supernates showed significantinhibition of mouse GIP when diluted 1:20.

To demonstrate that the monoclonal antibodies were specific to GIP, thesupernates from the five hybridomas that scored positive in thesuspension assay were diluted 1:1 with a 0.1 nM solution of humanglucagon-like peptide-1 (GLP-1). The mixtures were then added to LGLP-1Rcells. LGLP-1R cells are identical to LGIPR2 cells except they expressthe GLP-1 receptor and not the GIPR. The cells were incubated for 4hours at 37° C. before the mixtures were removed, the cells washed and62 -galactosidase content was assayed. None of the supernates inhibitednative GLP-1, indicating specificity to GIP.

Next, the two hybridomas that produced supernates that showedsignificant inhibition of mouse GIP when diluted 1:20 were expanded and˜5×10⁵ cells were harvested and total RNA was prepared using a kitpurchased from Ambion (RNaqueous-4PCR, Life Technologies). Twomicrograms of the total RNA was used to make first-strand cDNA using theSuprscript III first-strand system for cDNA synthesis purchased fromLife Technologies. The resultant cDNA was used to amplify the cDNAencoding the heavy and light chain variable sequences in two separatepolymerase chain reactions (PCRs).

To amplify the heavy chain variable sequences, an oligonucleotide withthe sequence CAGTCGAAGC TTTGAGGAGA CGGTGACCGTG GTCCCTTGGC CCCAG (SEQ IDNO: 43) was used as the reverse primer, and an oligonucleotide with thesequence: CAACTAGGAT CCAGGTSMAR CTGCAGSAGT CWGG (SEQ ID NO: 44) was usedas the forward primer. The reverse primer contains the sequence AAGCTT(SEQ ID NO: 45) at its 5′-end, which is the recognition sequence for therestriction enzyme HindIII. The forward primer contains the sequenceGGATCC (SEQ ID NO: 46) at its 5′-end, which is the recognition sequencefor the restriction enzyme BamHI. The resultant products of the PCRswere digested with the enzymes HindIII and BamHI, then ligated to theplasmid pUC18 which was also digested with the restriction enzymesHindIII and BamHI. The ligation reaction was performed using theFast-Link kit purchased from Epicentre. The ligation reaction was usedto transform E.coli DH5α bacterial cells (Life Technologies). Bacteriathat took up plasmid were selected on agar plates containing 50microgram/milliliter (μg/ml) carbenicillin. Colonies that grew on thecarbenicillin agar plates were picked and grown in 2 ml cultures for 16hours. The bacteria was harvested and plasmid DNA was isolated using thealkaline lysis mini-prep method. Purified plasmid DNA was digested withthe restriction enzymes BamHI and HindIII before the DNA was resolved byelectrophoresis through a 1.2% agarose gel in a Tris-borate buffer. DNAfragments in the gel were stained with ethidium bromide and visualizedusing an ultraviolet lamp. Plasmids that generated restriction fragmentswith approximate molecular sizes of 374 base pairs were sequenced usinga service purchased from Eurofins MWG Operon (Huntsville, Ala.).

To amplify the light chain variable sequences, the same procedure wasgenerally followed as described above. The main differences were that anoligonucleotide with the sequence: CAGTCGAAGC TTGTTAGATC TCCAGCTTG GTCCC(SEQ ID NO: 47) was used as the forward primer, and an oligonucleotidewith the sequence CAACTAGGAT CCGACATTCA GCTGACCCAG TCTCCA (SEQ ID NO:48) was used as the reverse primer. Again, the forward primer containsthe recognition sequence for HindIII (SEQ ID NO: 45), and the reverseprimer contains the recognition sequence for BamHI (SEQ ID NO: 46).Plasmids that generated restriction fragments with approximate molecularsizes of 355 base pairs were sequenced using a service purchased fromEurofins MWG Operon (Huntsville, Ala.).

The resulting mAbs identified using the procedure described above aremouse antibodies. These mouse antibodies were partially humanized byforming a chimeric antibody possessing the variable domains of the mouseheavy and light chains fused to human heavy and light chain constantregions, respectively. This was done by amplifying the heavy chain andlight chain variable sequences using the polymerase chain reaction(PCR). The templates used in the PCRs were the pUC18 derivativescontaining the corresponding variable heavy or variable light chain cDNAsequences. The heavy chain variable regions were amplified using anoligonucleotide with the sequence TCACGAATTC TCAGGTCCAG CTGCAGGAGT (SEQID NO: 49) as the forward primer, and an oligonucleotide with thesequence TTGGTGCTAG CTGAGGAGAC GGTGACCGT (SEQ ID NO: 50), as the reverseprimer. The forward primer contains the sequence GAATTC (SEQ ID NO: 51)at its 5′-end, which is the recognition sequence for the restrictionenzyme EcoRl. The reverse primer contains the sequence GCTAGC (SEQ IDNO: 52) at its 5′-end, which is the recognition sequence for therestriction enzyme Nhel. After PCR amplification, the DNA fragments weredigested with EcoRI and NheI and ligated to the plasmid pFUSEss-CHlg-hG1which was also digested with the restriction enzymes EcoRI and NheI. Theplasmid pFUSEss-CHlg-hG1 is a mammalian expression vector purchase fromInvivoGen (San Diego, Calif.). Cloning the variable heavy chain cDNAsequences into this plasmid produces a gene that encodes a chimericheavy chain consisting of the mouse variable region and a human constantregion. The ligation reaction was performed using the Fast-Link kitpurchased from Epicentre. The ligation reaction was used to transformE.coli DH5α bacterial cells (Life Technologies). Bacteria that took upplasmid were selected on agar plates containing 50 μg/ml zeomycin.Colonies that grew on the zeomycin agar plates were picked and grown in2 ml cultures for 16 hours. The bacteria were harvested and plasmid DNAwas isolated using the alkaline lysis mini-prep method. Purified plasmidDNA was digested with the restriction enzymes EcoRII and Nhel before theDNA was resolved by electrophoresis through a 1.2% agarose gel in aTris-borate buffer. DNA fragments in the gel were stained with ethidiumbromide and visualized using an ultraviolet lamp. Successful cloning wasconfirmed by identification of a DNA fragment with a molecular size of373 base pairs.

The light chain variable regions were amplified using an oligonucleotidewith the sequence GTCACGAATTCAGACATTCAGCTGACCCAG (SEQ ID NO: 55)containing the recognition sequence (SEQ ID NO: 51) for the restrictionenzyme EcoRI as the forward primer, and an oligonucleotide with thesequence AGCCACCGTA CGTTTGATCT CCAGCTTGGT CCCA (SEQ ID NO: 53) as thereverse primer. The reverse primer contains the sequence CGTACG (SEQ IDNO: 54) at its 5′-end, which is the recognition sequence for therestriction enzyme BsiWI. After PCR amplification, the DNA fragmentswere digested with EcoRI and Bswil and ligated to the plasmidpFUSE2ss-CLlg-hK which was also digested with the restriction enzymesEcoRI and Bswil. The plasmid pFUSE2ss-CLlg-hK is a mammalian expressionvector purchase from InvivoGen (San Diego, Calif.). Cloning the variablelight chain cDNA sequences into this plasmid produces a gene thatencodes a chimeric light chain consisting of the mouse variable regionand the human constant region. The ligation reaction was performed usingthe Fast-Link kit purchased from Epicentre. The ligation reaction wasused to transform E.coli DH5a bacterial cells (Life Technologies).Bacteria that took up plasmid were selected on agar plates containing 50μg/ml blastocidin. Colonies that grew on the blastocidin agar plateswere picked and grown in 2 ml cultures for 16 hours. The bacteria wereharvested and plasmid DNA was isolated using the alkaline lysismini-prep method. Purified plasmid DNA was digested with the restrictionenzymes EcoRII and BsiWI before the DNA was resolved by electrophoresisthrough a 1.2% agarose gel in a Tris-borate buffer. DNA fragments in thegel were stained with ethidium bromide and visualized using anultraviolet lamp. Successful cloning was confirmed by identification ofa DNA fragment with a molecular size of 353 base pairs.

To express chimeric antibodies containing the variable regions clonedfrom the hybridomas and human constant regions, pFUSEss-CHIg-hG1 and thepFUSE2ss-CLIg-hK derivatives containing the variable heavy chain andvariable light chain sequences, respectively, were introduced intoChinese hamster ovary (CHO-1) cells in culture. Plasmid DNA wasintroduced by transfection employing the cationic lipid reagentTurbofect (Thermo Scientific, Pittsburgh, Pa.) to facilitate the entryof DNA into cultured cells. Two days after transfection, cell supernatewas collected and analyzed for GIP-neutralizing activity. To demonstratethe ability of the chimeric mAbs to neutralize GIP, the cell supernatewas mixed with an equal volume of 2×10⁻⁹ M hGIP, before adding to LGIPR2cells in culture. After incubation at 37° C. for 4 hours, the cells werelysed and β-galactosidase activity assayed. In this assay, thecombination of the plasmids containing the variable light chainsequences and the variable heavy chain sequences from the hybridomadesignated 10g10, was able to neutralize GIP activity in the cell-basedreporter assay.

The variable regions of the mAbs made and identified using theprocedures disclosed above were maintained, and the constant regionswere substituted with human constant regions. Alternatively, the GIP mAbantagonists could be made using transgenic mice to make “fully” humanmAbs, or other technologies could be used as well. For example, theamino acids in the variable domains that are conserved in mouseantibodies could be replaced with amino acids that are conserved inhuman antibodies.

The resulting monoclonal antibody antagonist thus binds to GIP. Inparticular embodiments, the molecular antagonist used in thecompositions of the present disclosure can bind to an amino sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, and SEQ ID NO: 4. Put another way, the antagonist (e.g.monoclonal antibody) binds to epitopes contained in these foursequences.

In various embodiments, the molecular antagonist has a molecular weight(MW) of about 30 kDa to about 500 kDa, including from about about 120kDa to about 500 kDa. In other embodiments, the molecular antagonist hasa binding affinity for GIP characterized by an IC50 of about 0.1 nM toabout 7 nM.

As previously discussed, the monoclonal antibody antagonist that bindsto GIP has variable domains in the light chains and heavy chains thatbind to GIP, or more specifically the CDRs of the variable domains ofthe light chains and heavy chains bind to GIP. It is generallycontemplated that the molecular antagonists of the present disclosurecontain at least one complementarity determining region (CDR) that bindsto GIP. More specifically, the molecular antagonist contains at leastone CDR having at least 80% identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 31, SEQ ID NO: 32, and SEQID NO: 33. These CDRs were identified through various modifications ofthe variable domains in the light chain and heavy chain identified inthe 10g10 hybridoma referred to above. Those modifications are discussedin more detail in the Examples section below. More desirably, the CDR(s)has/have at least 85%, or at least 90%, or at least 95% identity, orhave 100% identity with these amino acid sequences. In more specificembodiments, the molecular antagonist contains two, three, four, five,or six CDRs having the requisite identity with two, three, four, five,or six different amino acid sequences in the above-mentioned group.

In some more specific embodiments, the molecular antagonist comprises alight chain variable domain having a first CDR and a second CDR, eachCDR having at least 95% identity to an amino acid sequence selected fromthe group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 23, and SEQ ID NO: 24. These CDRs were identified in the variabledomain of the light chain. More desirably, the CDRs have 100% identitywith these amino acid sequences.

In other particular embodiments, the molecular antagonist comprises alight chain variable domain having a first CDR with at least 80%identity to the amino acid sequence of SEQ ID NO: 20, a second CDR withat least 80% identity to the amino acid sequence of SEQ ID NO: 21, and athird CDR with at least 85% identity to the amino acid sequence of SEQID NO: 22. A variable domain containing the combination of these threeCDRs was identified as having a very high binding affinity for GIP. Moredesirably, the three CDRs have at least 85%, or at least 90%, or atleast 95% identity, or have 100% identity with these amino acidsequences.

In additional embodiments, the molecular antagonist comprises a heavychain variable domain having a first CDR with at least 80% identity tothe amino acid sequence of SEQ ID NO: 31, a second CDR with at least 80%identity to the amino acid sequence of SEQ ID NO: 32, and a third CDRwith at least 80% identity to the amino acid sequence of SEQ ID NO: 33.A variable domain containing the combination of these three CDRs wasidentified as having a very high binding affinity for GIP. Moredesirably, the three CDRs have at least 85%, or at least 90%, or atleast 95% identity, or have 100% identity with these amino acidsequences.

It is generally contemplated that these variable domains contain two orthree CDRs as specified above, with the CDRs being joined to each otherby linking groups. The linking groups can generally be any groups thatwill permit the CDRs to bind to GIP. For example, the linking groups canbe chains of amino acids, as are present in the natural variable domainsof antibodies. The amino acids can be of any desired length.

In particular embodiments, the molecular antagonist comprises a lightchain variable domain with at least 80% identity to an amino acidsequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, and SEQ ID NO: 19. More desirably, the light chainvariable domain has at least 85%, or at least 90%, or at least 95%, orhas 100% identity with one of these amino acid sequences. These variabledomains contain various combinations of the light chain CDRs of SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24,linked together with amino acids.

In other embodiments, the molecular antagonist comprises a heavy chainvariable domain with at least 80% identity to an amino acid sequenceselected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQID NO: 29, and SEQ ID NO: 30. More desirably, the heavy chain variabledomain has at least 85%, or at least 90%, or at least 95%, or has 100%identity with one of these amino acid sequences. These variable domainscontain various combinations of the heavy chain CDRs of SEQ ID NO: 31,SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, linked together withamino acids.

Also contemplated are molecular antagonists having any combination ofthe light chain variable domains and the heavy chain variable domainsdisclosed above. More specifically, some molecular antagonists containonly one light chain variable domain and only one heavy chain variabledomain. Others contain multiple light chain variable domains and heavychain variable domains; in these embodiments usually the multiple lightchain variable domains are the same, and the multiple heavy chainvariable domains are the same.

In some specific embodiments, the molecular antagonist comprises a lightchain variable domain and a heavy chain variable domain. The light chainvariable domain comprises a first CDR and a second CDR, each CDR havingat least 95% identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, and SEQ ID NO: 24; and the heavy chain variable domain comprises afirst CDR with at least 80% identity to the amino acid sequence of SEQID NO: 31, a second CDR with at least 80% identity to the amino acidsequence of SEQ ID NO: 32, and a third CDR with at least 80% identity tothe amino acid sequence of SEQ ID NO: 33. The light chain variabledomain may have 100% identity with one of the listed amino acidsequences. More particularly, the heavy chain variable domain has atleast 85%, or at least 90%, or at least 95%, or has 100% identity withone of the listed amino acid sequences.

In other specific embodiments, the molecular antagonist comprises alight chain variable domain and a heavy chain variable domain. The lightchain variable domain has at least 80% identity to an amino acidsequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, and SEQ ID NO: 19; and the heavy chain variabledomain has at least 80% identity to an amino acid sequence selected fromthe group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, andSEQ ID NO: 30. More particularly, the light chain variable domain and/orthe heavy chain variable domain has at least 85%, or at least 90%, or atleast 95%, or has 100% identity with one of their listed amino acidsequences.

In even more specific embodiments, the molecular antagonist has a lightchain variable domain having at least 80% identity to SEQ ID NO: 18, anda heavy chain variable domain having at least 80% identity to an aminoacid sequence selected from the group consisting of SEQ ID NO: 28, SEQID NO: 29, and SEQ ID NO: 30. Again, these domains can have at least85%, or at least 90%, or at least 95%, or 100% identity with one oftheir listed amino acid sequences.

In particularly desirable embodiments, the molecular antagonist has alight chain variable domain having at least 90% identity to SEQ ID NO:18, and a heavy chain variable domain having at least 90% identity toSEQ ID NO: 29. In more specific embodiments, these domains can have atleast 95%, or can have 100% identity with their listed amino acidsequence.

The molecular antagonists of the present disclosure have, in severaldifferent embodiments, a light chain variable domain and a heavy chainvariable domain as described above. It is particularly contemplated thatthe molecular antagonist could be a single-chain variable fragment(scFv), an F(ab′)₂ fragment, a Fab or Fab′ fragment, a diabody, atriabody, a tetrabody, or a monoclonal antibody of which these variabledomains would be a part.

A single-chain variable fragment (scFv) includes a light chain variabledomain and a heavy chain variable domain, joined together with a linkinggroup which usually has a length of about 10 to about 25 amino acids(though it does not need to be within this range). The N-terminus of onevariable domain is connected to the C-terminus of the other variabledomain. If desired, the scFV can be PEGylated (with polyethylene glycol)to increase its size, as with certolizumab pegol. Two scFvs can bejoined together with another linking group to produce a tandem scFv.

If a light chain variable domain and a heavy chain variable domain arejoined together with a shorter linking group to form an scFv, the twovariable domains cannot fold together, and the scFv will dimerize toform a diabody. Even shorter linking groups can result in the formationof trimers (i.e. a triabody) and tetramers (i.e. a tetrabody).

A whole monoclonal antibody is formed from two heavy chains and twolight chains. Again, each light chain and each heavy chain contains avariable domain. Each light chain is bonded to a heavy chain. The twoheavy chains are joined together at a hinge region. If the constantregion of the heavy chains are removed below the hinge region, anF(ab′)₂ fragment is produced which contains a total of four variabledomains. The F(ab′)₂ fragment can then be split into two Fab′ fragments.An Fab′ fragment contains sulfhydryl groups from the hinge region. A Fabfragment is formed when the constant region of the heavy chains isremoved above the hinge region, and does not sulfhydryl groups from thehinge region. However, all of these fragments contain a light chainvariable domain and a heavy chain variable domain.

In desirable embodiments explored in experiments described below, themolecular antagonist is a whole monoclonal antibody formed from lightchains and heavy chains having the variable regions / domains disclosedabove, combined with human constant regions. The constant region of theheavy chain can be any human isotype, including IgA1, IgA2, IgD, IgE,IgG1, IgG2, IgG3, IgG4, or IgM. The human constant region of the lightchain can be the kappa or lambda isotype. In specific embodiments, theheavy chain constant region is the IgG1 isotype, and the light chainconstant region is the kappa isotype.

In particular embodiments, the molecular antagonist is a monoclonalantibody with a light chain variable domain having at least 80% identityto SEQ ID NO: 18, and a heavy chain variable domain having at least 80%identity to an amino acid sequence selected from the group consisting ofSEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30. These domains can haveat least 85%, or at least 90%, or at least 95%, or 100% identity withone of their listed amino acid sequences.

In other particular embodiments, the molecular antagonist is a wholemonoclonal antibody with a light chain variable domain having at least90% identity to SEQ ID NO: 18, and a heavy chain variable domain havingat least 90% identity to SEQ ID NO: 29. These domains can have at least95%, or can have 100% identity with their listed amino acid sequence.

The molecular antagonist of GIP, which in particular forms is amonoclonal antibody, can then be used in a composition that can beadministered to a person. The composition contains a pharmaceuticallyeffective amount of the molecular antagonist of GIP. In particularembodiments, the composition contains the molecular antagonist in anamount of from about 0.1 to about 1000 milligrams per milliter of thecomposition (w/v).

The pharmaceutical compositions containing the molecular antagonist ofGIP is generally administered by a parenteral (i.e. subcutaneously,intramuscularly, intravenously, intraperitoneally, intrapleurally,intravesicularly or intrathecally) route, as necessitated by choice ofdrug and disease. The dose used in a particular formulation orapplication will be determined by the requirements of the particularstate of disease and the constraints imposed by the characteristics ofcapacities of the carrier materials. It contemplated in that in the mostdesirable form, the composition will be administered intravenously,intraperitoneally, or subcutaneously.

The pharmaceutical composition may include a pharmaceutically acceptablecarrier. The carrier acts as a vehicle for delivering the molecularantagonist. Examples of pharmaceutically acceptable carriers includeliquid carriers like water, oil, and alcohols, in which the molecularantagonists can be dissolved or suspended.

The pharmaceutical composition may also include excipients. Particularexcipients include buffering agents, surfactants, preservative agents,bulking agents, polymers, and stabilizers, which are useful with thesemolecular antagonists. Buffering agents are used to control the pH ofthe composition. Surfactants are used to stabilize proteins, inhibitprotein aggregation, inhibit protein adsorption to surfaces, and assistin protein refolding. Exemplary surfactants include Tween 80, Tween 20,Brij 35, Triton X-10, Pluronic F127, and sodium dodecyl sulfate.Preservatives are used to prevent microbial growth. Examples ofpreservatives include benzyl alcohol, m-cresol, and phenol. Bulkingagents are used during lyophilization to add bulk. Hydrophilic polymerssuch as dextran, hydroxyl ethyl starch, polyethylene glycols, andgelatin can be used to stabilize proteins. Polymers with nonpolarmoieties such as polyethylene glycol can also be used as surfactants.Protein stabilizers can include polyols, sugars, amino acids, amines,and salts. Suitable sugars include sucrose and trehalose. Amino acidsinclude histidine, arginine, glycine, methionine, proline, lysine,glutamic acid, and mixtures thereof. Proteins like human serum albumincan also competitively adsorb to surfaces and reduce aggregation of theprotein-like molecular antagonist. It should be noted that particularmolecules can serve multiple purposes. For example, histidine can act asa buffering agent and an antioxidant. Glycine can be used as a bufferingagent and as a bulking agent.

The pharmaceutical composition may be in the form of a powder,injection, solution, suspension, or emulsion. It is contemplated thatthe composition will be delivered by injection. Sometimes, the molecularantagonist of GIP can be lyophilized using standard techniques known tothose in this art. The lyophilized antagonist may then be reconstitutedwith, for example, suitable diluents such as normal saline, sterilewater, glacial acetic acid, sodium acetate, combinations thereof and thelike.

Dose will depend on a variety of factors, including the therapeuticindex of the drugs, disease type, patient age, patient weight, andtolerance. The dose will generally be chosen to achieve serumconcentrations from about 0.1 μg/ml to about 100 μg/ml in the patient.The dose of a particular patient can be determined by the skilledclinician using standard pharmacological approaches in view of the abovefactors. The response to treatment may be monitored by analysis of bloodor body fluid levels of glucose or insulin levels, or by monitoring fatlevels in the patient. The skilled clinician will adjust the dose basedon the response to treatment revealed by these measurements. A singleadministration may usually be sufficient to produce a therapeuticeffect, but it is contemplated that multiple administrations will beused to assure continued response over a substantial period of time.Because of the protein-like nature of the molecular antagonistsdisclosed herein, it is believed that the antagonists will have a longhalf-life in the body, so that the composition will only need to beadministered once or twice a month, or possibly once a week. Thecirculating concentration of the molecular antagonist should besufficient to neutralize GIP that is generated during and after eating.

The pharmaceutical compositions containing the GIP molecular antagonistsof the present disclosure can be used to treat ischemic heart diseaseand other clinical manifestations associated with ischemic heartdisease. The term “treat” is used to refer to a reduction in progressionof the disease, a regression in the disease, and/or a prophylactic usageto reduce the probability of presentation of the disease. It is believedthat the GIP molecular antagonists will inhibit binding of GIP to itsreceptor in cardiomyocytes, which may have a cardioprotective effect, orthat cardioprotective effects may arise indirectly through inhibition ofGIP binding elsewhere in the body.

The present disclosure will further be illustrated in the followingnon-limiting three sets of working examples, it being understood thatthese examples are intended to be illustrative only and that thedisclosure is not intended to be limited to the materials, conditions,process parameters and the like recited herein. All proportions are byweight unless otherwise indicated.

EXAMPLES Example 1

Monoclonal antibodies were generated and screened as described aboveusing hybridomas to identify a monoclonal antibody having high bindingaffinity for gastric inhibitory peptide (GIP) in suspension. The 10g10monoclonal antibody was thus identified.

The 10g10 light chain variable domain has the amino acid sequence of SEQID NO: 16. The first CDR of the light chain variable domain has theamino acid sequence of SEQ ID NO: 25. The second CDR of the light chainvariable domain has the amino acid sequence of SEQ ID NO: 26. The thirdCDR of the light chain variable domain has the amino acid sequence ofSEQ ID NO: 22. cDNA encoding for the 10g10 light chain variable domainhas the nucleotide sequence of SEQ ID NO: 35.

The 10g10 heavy chain variable domain has the amino acid sequence of SEQID NO: 27. The first CDR of the heavy chain variable domain has theamino acid sequence of SEQ ID NO: 31. The second CDR of the heavy chainvariable domain has the amino acid sequence of SEQ ID NO: 34. The thirdCDR of the heavy chain variable domain has the amino acid sequence ofSEQ ID NO: 33. cDNA encoding for the 10g10 heavy chain variable domainhas the nucleotide sequence of SEQ ID NO: 39.

For comparison, the 14B9 antibody was identified as not having bindingaffinity for GIP. The 14B9 light chain variable domain has the aminoacid sequence of SEQ ID NO: 7. The first CDR of the light chain variabledomain has the amino acid sequence of SEQ ID NO: 9. The second CDR ofthe light chain variable domain has the amino acid sequence of SEQ IDNO: 26. The third CDR of the light chain variable domain has the aminoacid sequence of SEQ ID NO: 10. cDNA encoding for the 14B9 light chainvariable domain has the nucleotide sequence of SEQ ID NO: 8.

The 14B9 heavy chain variable domain has the amino acid sequence of SEQID NO: 11. The first CDR of the heavy chain variable domain has theamino acid sequence of SEQ ID NO: 13. The second CDR of the heavy chainvariable domain has the amino acid sequence of SEQ ID NO: 14. The thirdCDR of the heavy chain variable domain has the amino acid sequence ofSEQ ID NO: 15. cDNA encoding for the 14B9 heavy chain variable domainhas the nucleotide sequence of SEQ ID NO: 12.

Example 2

Materials and Methods

30 nine-week old male C57BL/6 mice were purchased from JacksonLaboratories (Bar Harbor, Me.). On the first day of the study, eachmouse weighed between 19 and 25 grams. The mice were then subsequentlydivided at random into three groups of 10 mice each.

All mice had free access to food and water throughout the study. Themice were all housed in an animal facility kept at 22±2° Celsius with a12-hour light/12-hour dark cycle. Mice were housed in groups of five percage until they each reached a weight of 25 grams. Mice were thensubsequently housed two to three animals per cage.

A high-fat diet (HFD) (catalog no. TD.06414) and a low-fat isocaloricdiet (catalog no. TD.08806) were purchased from Harlan-Teklad(Indianapolis, Ind.). The HFD consisted, by weight, of approximately23.5% protein, 27.3% carbohydrates and 34.3% fat. The HFD provided 18.4%of total calories from protein, 21.3% from carbohydrates, and 60.3% fromfat, and 5.1 kcal/gram. The isocaloric diet consisted, by weight, ofapproximately 18.6% protein, 62.6% carbohydrates and 4.2% fat. Theisocaloric diet provided 20.5% of total calories from protein, 69.1%from carbohydrates, and 10.4% from fat, and 3.6 kcal/gram.

One group of 10 mice (HFD-control group) was fed the HFD for 17 weeksand administered 0.1 mL phosphate buffered saline (PBS) byintraperitoneal (i.p.) injection five times per week.

The second group of 10 mice (HFD-mAb group) was fed the HFD for 17 weeksand administered the 10g10 GIP mAb in PBS five times per week. On Mondaythrough Thursday, a 0.1 mL solution consisting of 0.2 mg/mL mAb in PBSwas administered by i.p. injection. On each Friday, a 0.1 mL solutionconsisting of 0.4 mg/mL mAb in PBS was administered by i.p. injection.This resulted in administration of 10 mg/kg BW of the GIP mAb on fourdays of the week and 20 mg/kg BW of the GIP mAb on one day of the week,for each week of the study. This dosing regimen was continued over thecourse of the study.

The third group of 10 mice (iso-diet group) was fed the isocaloric dietfor 17 weeks. No injections were administered to this group.

After 17 weeks on the special diets, mice in the three groups weresacrificed. Whole blood was collected, serum was prepared, and thelevels of many different components were measured.

Results

FIG. 1 shows triglyceride (TG) levels measured in the sera of each groupof mice. TG levels in HFD-control mice were 1.44 times greater than TGlevels in HFD-mAb mice. Higher TG levels are undesirable.

FIG. 2 shows average total cholesterol (TC) levels measured in the seraof each group of mice. TC levels in HFD-control mice were 1.55 timesgreater than TC levels in HFD-mAb mice. Higher TC levels areundesirable.

FIG. 3 shows the average ratio of high-density lipoprotein (HDL) tototal cholesterol (TC) measured in the sera of each group of mice. Theratio of HDL to total cholesterol in HFD-control mice was 20% lower thanthe ratio of HDL to total cholesterol levels in HFD-mAb mice. Lowerratios of HDL to total cholesterol levels are undesirable.

FIG. 4 shows average low-density lipoprotein (LDL) levels measured inthe sera of each group of mice. LDL levels in HFD-control mice were 1.95times greater than LDL levels in HFD-mAb mice. Higher LDL (the “bad”cholesterol) levels are undesirable.

Example 3 Modification of Antibodies

The light chain variable region of the 10g10 mAb was further modified bysubstituting specific amino acids conserved in mouse antibodies withspecific amino acids conserved in human antibodies. This was done to“humanize” the light chain variable region to make it more similar to ahuman variable region, and increase the chance that the human immunesystem would not recognize the “humanized” mAb as a foreign substance.To humanize the light chain variable region, oligonucleotides werechemically synthesized. The sequences of the oliogonucleotides weresimilar to the sequence of the 10g10 mAb light chain variable region,except specific bases were changed. The base pairs changed werepredicted to change specific amino acids of the light chain variableregion when the sequence was translated into protein. A DNA synthesisreaction was used to produce double-stranded (ds) DNA. Three differentds-oligonucleotides were synthesized and then digested with therestriction endonucleases HindIII and NotI, before being ligated into amammalian expression vector. The mammalian expression vector contained asignal sequence and the constant region of a human light chain. Theds-oligonucleotides were ligated to the vector in such a way that thevariable region and the constant region, when expressed as mRNA and thentranslated into protein, would generate an antibody light chain.

The three resulting humanized light chains are referred to as LC1, LC2,and LC3 herein. The amino acid sequence of LC1 is SEQ ID NO: 17, and thecDNA sequence for LC1 is SEQ ID NO: 36. The amino acid sequence of LC2is SEQ ID NO: 18, and the cDNA sequence for LC1 is SEQ ID NO: 37. Theamino acid sequence of LC3 is SEQ ID NO: 19, and the cDNA sequence forLC1 is SEQ ID NO: 38.

The heavy chain variable region of the 10g10 mAb was also furthermodified in the same manner. Again, the base pairs changed werepredicted to change specific amino acids of the heavy chain variableregion when the sequence was translated into protein. Three differentds-oligonucleotides for the chain were synthesized and then ligated intoa mammalian expression vector. The mammalian expression vector containeda signal sequence and the constant region of a human heavy chain. Theds-oligonucleotides were ligated to the vector in such a way that thevariable region and the constant region, when expressed as mRNA and thentranslated into protein, would generate an antibody heavy chain.

The three resulting humanized heavy chains are referred to as HC1, HC2,and HC3 herein. The amino acid sequence of HC1 is SEQ ID NO: 28, and thecDNA sequence for HC1 is SEQ ID NO: 40. The amino acid sequence of HC2is SEQ ID NO: 29, and the cDNA sequence for HC1 is SEQ ID NO: 41. Theamino acid sequence of HC3 is SEQ ID NO: 30, and the cDNA sequence forHC1 is SEQ ID NO: 42.

Monoclonal antibodies (mAbs) containing the humanized light chainvariable regions and heavy chain variable regions were produced in CHOcells. A mammalian vector possessing the coding sequence for an antibodylight chain was co-transfected with a mammalian vector possessing thecoding sequence for an antibody heavy chain. A total of 9 uniquecombinations were transfected into CHO cells: LC1/HC1, LC1/HC2, LC1/HC3,LC2/HC1, LC2/HC2, LC2/HC3, LC3/HC1, LC3/HC2, and LC3/HC3. Monoclonalantibodies derived from these combinations were compared to the 10g10mAb.

CHO cells co-transfected with the mammalian expression vectors weregrown in culture and the supernate collected. The supernate wasevaluated for the presence of GIP-binding mAbs by direct binding ELISA.Human GIP was fixed to the bottom of the wells on 96-well plates. Serialdilutions of the supernate were added to different wells of the 96-wellplate. After a one hour incubation, the wells were washed twice with PBSbefore a secondary antibody that specifically binds to human IgG wasadded to each well. The antibody that specifically binds to human IgGwas conjugated with the enzyme horse radish peroxidase (HRP). Afterincubation for one hour, the wells were washed with PBS before the HRPsubstrate was added. If HRP was present in a well, the substrate wasconverted to a colored compound. The amount of color intensity was readusing a spectrophotometer. The amount of color produced was proportionalto the amount of HRP-antibody conjugate remaining in each well. Theamount of color produced was also proportional to the amount of mAbbound to hGIP in each well.

The results of this evaluation indicated that the various combinationsof the light chain / heavy chain vectors bound human GIP in thefollowing order (highest binding affinity to lowest binding affinity):LC2/HC1, LC2/HC2, LC3/HC2, LC1/HC2, LC2/HC3, LC3/HC1, LC1/HC1, 10g10,LC1/HC3, and LC3/HC3.

Antibodies produced in 100 ml suspension cultures of CHO cellsco-transfected with the combination of expression vectors of LC2/HC1,LC2/HC2, and LC2/HC3 were purified using Protein A agarose. Each lightchain (LC1, LC2, LC3) and each heavy chain (HC1, HC2, HC3) alsoindividually displayed the ability to specifically bind to GIP.

Next, the ability of these three purified mAbs to neutralize hGIP andprevent ligand-receptor interaction, receptor activation andreceptor-dependent signaling was tested using a cell culture system.This system used reporter cells (LGIPR3a cells), which possess the lacZgene under the control of a cyclic adenosine monophosphate(cAMP)-responsive promoter and expresses the rat GIPR on the cellsurface. The addition of GIP to these cells leads to activation of theGIPR, induction of a signaling cascade that leads to accumulation ofcAMP, induction of the lacZ gene and synthesis of β-galactosidase. Afterthe addition of a test sample and incubation for 4 hours, a colorimetricassay was used to measure β-galactosidase content in cell lysates. Thedegree of color change was proportional to the level of β-galactosidaseactivity. The level of β-galactosidase activity was dependent on theamount of free biologically active GIP in the test sample.

The mAbs were mixed with a solution of human GIP (hGIP) in DMEM media atfinal concentrations of 25 g/ml for the mAb and 0.1 nM for hGIP. Themixtures were then added to LGIPR3a cells and incubated before washingand assaying for β-galactosidase. The results are shown in FIG. 5. 10 g10 represents the original mouse mAb. The GIP acted as a positivecontrol. Fc represents purified human Fc fragment, which acted as anegative control.

The results showed that the humanized antibodies neutralized hGIP invitro similar to the 10g10 mouse mAb. The LC2/HC2 humanized antibody wasthe most effective of the three humanized mAbs. It was also moreeffective than the original mouse mAb. A lower relative activity isdesired here, and the LC2/HC2 had a relative activity of 0.0035, versus0.0055 for the 10g10 mouse mAb.

The complementarity determining regions (CDRs) of the variable regionsof the six humanized light chains (LC1, LC2, LC3) and heavy chains (HC1,HC2, HC3) was also determined. LC1 had the CDRs of SEQ ID NO: 20, SEQ IDNO: 23, and SEQ ID NO: 22. LC2 had the CDRs of SEQ ID NO: 20, SEQ ID NO:21, and SEQ ID NO: 22. LC3 had the CDRs of SEQ ID NO: 20, SEQ ID NO: 24,and SEQ ID NO: 22. HC1, HC2, and HC3 all had the same CDRs, which wereSEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.

Example 4

In separate studies, the LC2/HC2 humanized antibody (mAbH) was comparedto the 10g10 antibody (mAb_(m)). Binding affinity was determined bysurface plasmon resonance, and a reporter cell line expressing GIPreceptors was used in a modified Schild's assay to demonstratemAb-dependent GIP neutralization in vitro. Finally, the GIP mAbH (10 or30 mg/kg BW) was administered to C57BL/6 mice intraperitoneally. Bloodsamples were collected immediately before injection (time 0) and attimes 1, 2 4, 8 and 12 hours after injection and also 1, 2, 4, 7 and 14days after injection. Blood was diluted in 1 mM EDTA and centrifuged for1 minute at 20,000 gravities to remove cells and the diluted plasmasamples were assayed for bioavailable humanized anti-GIP mAb using aspecific ELISA. The C_(max) and T_(1/2) were determined, and the resultsare shown in FIG. 6. For each time point, the average of 4 mice±SE ispresented. T_(1/2) is about nine days.

In separate studies, mice fasted for 1 hour were administered either 30mg/kg BW mAbH or control vehicle (PBS) intraperitoneally. The mice werethen fasted for an additional 5 hours before glucose (1.5 g/kg BW) wasgiven by oral gavage, and blood was collected at 0 and 15 minutes; theprotocol was repeated on the mice 1 week and 2 weeks later. Plasmainsulin was measured by ELISA.

Similar to the mAbm, the mAbH neutralized GIP signaling in vitro in aconcentration-dependent manner, and the Schild's assay plot showed mAbHand mAbm equilibrium dissociation constants of 2.2 μM and 3.2 μM,respectively, indicating similarly that the mAbH binds more strongly toGIP than does the mAbm. The measured plasma C_(max) values for the 10and 30 mg/kg BW doses of GIP mAbH were 10.1±0.7 μg/ml and 32.6±1.0μg/ml, respectively. The GIP mAbH inhibited the 15-minute insulinresponses to oral glucose at 5 hours, 1 week, and 2 weeks aftertreatment by 47±21% (P=0.01), 45±21% (P=0.01), and 13.8±31%. The GIPmAbH was shown to bind GIP with a higher affinity than the mAbm, andsimilarly it inhibits GIP signaling in vitro more potently than the10g10 mAb_(m). Furthermore, the calculated T_(1/2) for the GIP mAb_(H)in vivo is comparable to other biological agents currently in use.

Example 5

The LC2/HC2 humanized antibody (mAb_(H)) and the 10g10 antibody (mAbm)are administered (30 mg/kg BW) to mice after experimental myocardialinfarction by permanent occlusion of the left anterior descendingartery, and compared to control mice to whom no antibody isadministered. The mAbH mice survive with a much lower mortality ratethan both the mAb_(m) mice and the control mice.

The LC2/HC2 humanized antibody (mAb_(H)) and the 10g10 antibody(mAb_(m)) are administered (30 mg/kg BW) to mice after experimentalheart failure produced by injection of doxorubicin (20 mg/kg BW), andcompared to control mice to whom no antibody is administered. ThemAb_(H) mice survive with a much lower mortality rate than both the mAbmmice and the control mice.

While particular embodiments have been described, alternatives,modifications, variations, improvements, and substantial equivalentsthat are or may be presently unforeseen may arise to applicants orothers skilled in the art. Accordingly, the appended claims as filed andas they may be amended are intended to embrace all such alternatives,modifications, variations, improvements, and substantial equivalents.

1. A method of treating ischemic heart disease or clinicalmanifestations thereof, comprising: administering to a person acomposition comprising a pharmaceutically effective amount of amolecular antagonist of gastric inhibitory polypeptide (GIP), whereinthe molecular antagonist comprises at least one complementaritydetermining region (CDR) with at least 80% identity to an amino acidsequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 31, SEQ IDNO: 32, and SEQ ID NO:
 33. 2. The method of claim 1, wherein themolecular antagonist comprises a light chain variable domain having afirst CDR and a second CDR, each CDR having at least 95% identity to anamino acid sequence selected from the group consisting of SEQ ID NO: 20,SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO:
 24. 3. Themethod of claim 1, wherein the molecular antagonist comprises a lightchain variable domain having a first CDR with at least 80% identity tothe amino acid sequence of SEQ ID NO: 20, a second CDR with at least 80%identity to the amino acid sequence of SEQ ID NO: 21, and a third CDRwith at least 85% identity to the amino acid sequence of SEQ ID NO: 22.4. The method of claim 1, wherein the molecular antagonist comprises alight chain variable domain having at least 80% identity to an aminoacid sequence selected from the group consisting of SEQ ID NO: 16, SEQID NO: 17, SEQ ID NO: 18, and SEQ ID NO:
 19. 5. The method of claim 1,wherein the molecular antagonist comprises a heavy chain variable domainhaving a first CDR with at least 80% identity to the amino acid sequenceof SEQ ID NO: 31, a second CDR with at least 80% identity to the aminoacid sequence of SEQ ID NO: 32, and a third CDR with at least 80%identity to the amino acid sequence of SEQ ID NO:
 33. 6. The method ofclaim 1, wherein the molecular antagonist comprises a heavy chainvariable domain having at least 80% identity to an amino acid sequenceselected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQID NO: 29, and SEQ ID NO:
 30. 7. The method of claim 1, wherein themolecular antagonist comprises a light chain variable domain and a heavychain variable domain; wherein the light chain variable domain comprisesa first CDR and a second CDR, each CDR having at least 95% identity toan amino acid sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24; andwherein the heavy chain variable domain comprises a first CDR with atleast 80% identity to the amino acid sequence of SEQ ID NO: 31, a secondCDR with at least 80% identity to the amino acid sequence of SEQ ID NO:32, and a third CDR with at least 80% identity to the amino acidsequence of SEQ ID NO:
 33. 8. The method of claim 1, wherein themolecular antagonist is a single-chain variable fragment (scFv), anF(ab′)₂ fragment, a Fab or Fab′ fragment, a diabody, a triabody, atetrabody, or a monoclonal antibody.
 9. The method of claim 1, whereinthe molecular antagonist comprises a light chain variable domain and aheavy chain variable domain; wherein the light chain variable domain hasat least 80% identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ IDNO: 19; and wherein the heavy chain variable domain has at least 80%identity to an amino acid sequence selected from the group consisting ofSEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO:
 30. 10. Themethod of claim 9, wherein the molecular antagonist is a monoclonalantibody with a light chain variable domain having at least 80% identityto SEQ ID NO: 18, and a heavy chain variable domain having at least 80%identity to an amino acid sequence selected from the group consisting ofSEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO:
 30. 11. The method of claim9, wherein the molecular antagonist is a whole monoclonal antibody witha light chain variable domain having at least 90% identity to SEQ ID NO:18, and a heavy chain variable domain having at least 90% identity toSEQ ID NO:
 29. 12. The method of claim 1, wherein the molecularantagonist binds to an amino acid sequence of GIP, the amino acidsequence being selected from the group consisting of SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, and SEQ ID NO:
 4. 13. The method of claim 1,wherein the molecular antagonist is a monoclonal antibody comprisinghuman constant regions.
 14. The method of claim 1, wherein the molecularantagonist has a molecular weight of about 30 kDa to about 500 kDa. 15.The method of claim 1, wherein the molecular antagonist has a bindingaffinity for GIP characterized by an IC₅₀ of about 0.1 nM to about 7 nM.16. The method of claim 1, wherein the composition is administeredintravenously, intraperitoneally, or subcutaneously.
 17. The method ofclaim 1, wherein the composition further comprises a pharmaceuticalexcipient selected from the group consisting of buffering agents,surfactants, preservative agents, bulking agents, polymers, andstabilizers.
 18. The method of claim 1, wherein the composition is inthe form of a powder, injection, solution, suspension, or emulsion. 19.The method of claim 1, wherein the composition contains the molecularantagonist in an amount of from about 0.1 to about 1000 milligram permilliliter of the composition.
 20. The method of claim 1, wherein thecomposition is lyophilized; or wherein the clinical manifestationsthereof related to ischemic heart disease include stable anginapectoris, unstable angina, acute coronary syndrome; ST elevationmyocardial infarction, non-ST elevation myocardial infarction, andunstable angina.